The invention particularly relates to membranes which can, for example, be used for culturing adherent cells of various types. Like most cells in vivo, many cells are adherent cells, or anchorage-dependent cells; that is, they can metabolize and divide only if they are attached to a surface or substratum. Only cells of the circulatory system (e.g., lymphocytes and red blood cells) grow unattached and suspended in solution in vitro. While many anchorage-dependent cells may grow on glass or synthetic surfaces, these cells often lose their ability to differentiate and respond to hormones. The loss of cellular morphology not only entails a loss of function, but also prevents regenerative power in a longer-term culture system. Longer-term cultivation would however be of great significance, for example, with the use of human cells for tissue culture, and many cells are not available in any quantity. For this reason, such tissue culture dishes are often coated with extracellular matrix components such as collagen or fibronectin. However, the use of xenogenic factors is a clear disadvantage, especially if the cells as such or on a matrix as used for medical treatment of human beings, as it will bring along risks of contamination and may result in adverse reactions in the patient treated.
The failure of cells to grow on such surfaces or keep their abilities is, for example, a major limitation of current tissue culture techniques. Tissue cultures are a potential source of tissues and organs which could be used for transplantation into humans. For example, tissue cultured skin cells could potentially be used in skin grafts. The aim is to develop biological substitutes that can restore and maintain normal function, for example, by the use of acellular matrices, which will depend on the body's ability to regenerate for proper orientation and direction of new tissue growth, or by the use of matrices or membranes with cells adhered thereto (Atala (2006): Recent developments in tissue engineering and regenerative medicine. Curr. Opin. Pediatr. 16, 167-171). Cells can also be used for therapy via injection, either with carriers or alone. In such cases, the cells need to be expanded in culture, attached to a support matrix, and then reimplanted into the host after expansion. Veterinary therapeutic applications are available today and may represent an additional application of membranes for cell cultivation.
The ability to culture cells, especially adherent cells, is important also because they represent biological “factories” capable of producing large quantities of bio products such as growth factors, antibodies and viruses. These products can then be isolated from the cell cultures and used, for example, to treat human diseases.
Cell cultures also are emerging tools for biocompatibility and toxicology studies in the field of pharmaceutical and life science industry.
Finally, tissue cultures usually comprise cells from only one or a few tissues or organs. Consequently, cell cultures provide scientists with a system for studying the properties of individual cell types without the complications of working with the entire organism.
A known method for adherent cell cultures involves a hollow fiber membrane bioreactor. In this system, the cells are generally attached to the lumen of a cylindrical hollow fiber membrane. Culture media and oxygen flow through the hollow fiber membrane. The molecular weight cut-off of the membrane permits nutrients and oxygen to reach the cells without allowing the cells to escape.
A variety of polymers has been suggested for producing semipermeable membranes for cell and tissue culture (US 2007/269489 A). They include polyalginate, polyvinylchloride, polyvinylidene fluoride, polyurethane isocyanate, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polysulfone, polyethersulfone, polystyrene, polyurethane, polyvinyl alcohol, polyacrylonitrile, polyamide, polymethylmethacrylate, polytetrafluoroethylene, polyethylene oxide and combinations of such polymers. The polymeric support may also consist of polyethylene terephthalate (PET) or polycarbonate. Further materials which were suggested, for example, as scaffolds for transplantable tissue material, are cellulose, macroporous collagen carriers, or biodegradable matrices.
EP 0 362 588 A1 describes the preparation of membranes which provide for a high concentration of immobilization sites for cells or other bioactive ingredients. The membranes disclosed can be flat or hollow fiber membranes. They are polysulfone (PSf)-based membranes and may further comprise, for example, polyethylene glycol, polyvinylpyrrolidone (PVP) or various polyurethane (PU) pre-polymers. Membranes specifically disclosed are made from polysulfone (Udel™ 3500) in concentrations of at least 8-35% in the casting solution and PVP, or polysulfone and an isocyanate-capped polyurethane prepolymer (BIOPOL™) in concentrations from 6% to 11% in the casting solution. The reference does not give any data on the actual possibility to use the membrane for supporting a cell culture of adherent cells.
WO 93/00439 A1 describes maintaining a cell culture within a biocompatible, semipermeable membrane in which the cells are stimulated and secrete active factor. The semipermeable membrane used permits the diffusion of the active factor therethrough while excluding detrimental agents present in the external environment from gaining access to the culture. The membrane described has a tubular shape and is said to enable the diffusion of molecules having a molecular weight of up to 150 kDa. Suggested materials for said membranes are acrylic copolymers, polyvinylchloride, polystyrene, polyurethane, polyamide, polymethacrylate, polysulfone, polyacrylate, polyvinylidene fluoride, polyurethaneisocyanate, polyalginate, cellulose acetate, polysulfone, polyvinyl alcohols, polyacrylonitrile, polyethylene oxide, and derivatives, and mixtures thereof. The membrane does not, as such, have to serve as a matrix for cell adhesion in this disclosure.
WO 90/11820 A2 discloses flat membranes with surfaces usable for cell growth in vitro or, as an artificial implant, in vivo. The membrane is described as being porous with a pore size in the range of from 0.1 to 100 microns and having a finger-like configuration in an intermediate layer. The membrane comprises a hydrophobic polymer and a hydrophilic polymer. Examples given for the hydrophobic polymer are polyurethane, polyether urethane, polyurethane urea, polyether urethane urea, polyvinylidene fluorides, polyvinyl fluoride, polysulfone, polyamides, polyethersulfone, polyesters, polycarbonates, preferably polyether urethane, and copolymers thereof. Examples given for the hydrophilic polymer are polyacrylic (or methacrylic) acids and co-, ter- and tetrapolymers, polyacrylic (or methacrylic) esters, polyacrylic (or methacrylic) salts, polyhydroxyethyl (or propyl) acrylate (or methacrylate) or hydroxypropyl or trishydroxypropyl acrylamide and co-, ter- and tetrapolymers, polydimethyl (or diethyl) amino ethyl acrylate (or methacrylate or amino propylmethacrylamide) and co-, ter- and tetrapolymers, polyacrylamide (or N-hydroxymethyl) acrylamide and co-, ter- and tetrapolymers, polyvinylpyrrolidone and co-, ter- and tetrapolymers, carboxymethyl cellulose and hydroxyethyl (or hydroxypropyl) cellulose. The reference specifically discloses a membrane made from polyether urethane and a copolymer of acrylic acid and ethyl hexyl methacrylate. The reference does not report whether or not it can be used for adhering and culturing cells.
U.S. Pat. No. 5,431,817 generically discloses membranes comprising as components polysulfone, polyethersulfone or polyestersulfone in an amount of at least from 8% to 35% of the casting solution, optionally further polymers such as PEG or PVP, and isocyanate end-capped polyurethane prepolymers in an amount of from 1% to 20% of the casting solution.
Membranes which are specifically disclosed are made from a polysulfone, a polyurethane prepolymer and PEG. The reference is silent on the ability of the membrane to be used as a matrix for the adherence and culturing of cells.
U.S. Pat. No. 5,151,227 also generically discloses membranes comprising as components polysulfone, polyethersulfone or polyestersulfone in an amount of at least from 8% to 35% of the casting solution, optionally further polymers such as PEG or PVP, and isocyanate end-capped polyurethane prepolymers. However, only membranes comprising polysulfone and PVP are specifically disclosed.
None of the references specifically discloses a membrane comprising, as a first component, polysulfone, polyethersulfone or polyarylethersulfone, PVP as a second component, and polyurethane as a third component. None of the references shows that the membranes disclosed therein can serve as a matrix for culturing adherent cells.
Apart from the problem of identifying membranes which could be used as a matrix for the cultivation of adherent cells, membranes currently known in the art suffer from their inability to sufficiently promote and sustain adherence, expansion, differentiation and extended life-span without the pre-treatment of said membranes or matrices, or the addition of exogenous factors, such as, for example, fibronectin, laminin or collagen.
For example, Fissell (2006) in Expert Rev. Med. Devices 3(2), 155, reviews efforts with regard to developing an artificial kidney based on adhering renal tubule cells to a synthetic polysulfone-based hollow-fiber membrane. In this case the membrane has to be coated with ProNectin-L™ in order to promote attachment of the cells.
U.S. Pat. No. 6,150,164 and U.S. Pat. No. 6,942,879 both present elaborate ways towards a bioartificial kidney based on renal cells such as, for example, endothelial cells or so-called renal stem cells, which are seeded into hollow fibers. Hollow fiber membranes which are mentioned as being useful are based on cellulose, polyacrylonitrile, polysulfone and other components or copolymers thereof. The internal and external surface of the hollow fiber is pre-coated with suitable extracellular matrix components (EMC) including Type I collagen, Type IV collagen, laminin, Matrigel, proteoglycan, fibronectin and combinations thereof. Only after such treatment the cells can be seeded.